The EGF receptor is a plasma membrane tyrosine kinase that is involved in the control of cell growth. Upon binding EGF, the EGF receptor dimerizes and transduces its signal via the activation of its intracellular tyrosine kinase. The EGF receptor associates with low density, cholesterol-enriched membrane domains, called rafts, that may represent a mechanism for organizing and regulating signal transduction. In the previous grant, we showed that alterations in cholesterol levels affect both EGF receptor binding and kinase activities, suggesting that the association of the EGF receptor with rafts may modulate receptor function. In addition, we developed a single-molecule method, fluorescence intensity cluster analysis (FICA), that allows us to define the distribution of the EGF receptor among clusters of increasing size on the plasma membrane in live cells. Using this method, we showed that cholesterol depletion enhances receptor clustering, suggesting the involvement of rafts in this process. We have also shown that EGF stimulation leads to the formation of clusters containing 4 or more receptors. However, receptor monomers still exist in cells stimulated with saturating doses of EGF. Results from a new enzyme complementation assay that assesses EGF receptor dimerization also suggest that EGF receptor clusters dissociate at high concentrations of EGF. The long term goal of the research in my laboratory is to understand the mechanism of activation of the EGF receptor and the possible contribution of membrane microdomains to this process. The specific aims of this new proposal are to: 1) Define the structural features of the membrane proximal portion of the extracellular domain of the EGF receptor that are involved in mediating high affinity EGF binding, signal transduction and association of the receptor with lipid rafts;2) Elucidate the molecular basis of EGF receptor clustering in response to EGF and changes in membrane cholesterol levels;and, 3) Determine the contribution of dimer dissociation to the process of EGF receptor activation. These studies will be accomplished using traditional mutagenesis analysis as well as our new method for brightness analysis, FICA, and our new enzyme complementation assay. The EGF receptor is over-expressed or constitutively activated in a variety of human tumors and several current therapies are based on inhibiting the function of the EGF receptor. To fully understand the process of signal transduction and to maximize our ability to target the EGF receptor for therapeutic purposes, it is necessary to establish a coherent model for the mechanism of activation of this receptor and to understand how the membrane environment of the receptor contributes to the overall regulation of EGF receptor function. The experiments proposed in this grant specifically address this need by examining a new model for activation of the EGF receptor and by further elucidating the potential involvement of cholesterol-rich membrane microdomains in modulating EGF receptor-mediated signaling.